OEA restored ethanol/THC-related decreases in both short-term spatial memory  and circulating levels of BDNF, reduced cell proliferation (Mki67 and IdU+ cells) and maturation , and improved cell survival (Casp3 and BrdU+ cells) in the dorsal hippocampus. Interestingly, OEA alone or combined with THC also decreased the mRNA levels of neurotrophic factors  and the NT3 receptor TrkC, but increased the BDNF receptor TrkB in the hippocampus of ethanol-exposed rats. These effects were likely associated with a OEA-specific phosphorylation of AKT and ERK1, key signaling regulators of cell proliferation and survival. These results suggest a regulatory role of OEA in short-term spatial memory and hippocampal neurogenesis through BDNF/AKT/ERK1 signaling in response to acute THC in an alcoholic context during adolescence.Oleoylethanolamide is an endogenous NAE that modulates ethanol-seeking behavior and ethanol-induced neuroinflammation. In the present study we further analyze the role of OEA in hippocampal neurogenesis, BDNF-ERK signaling, and spatial memory that are affected by alcohol. Additionally, we addressed the effects of OEA on the association of alcohol and cannabis, a frequent combination in human alcohol addicts, and whose long-term effects are far from being understood. To this end, OEA  was pharmacologically administered for 5 days/week in a preclinical model of adolescent rats with binge-like consumption (1 day/week) of ethanol  combined or not with acute administrations of Δ The excessive consumption of alcohol is associated with alterations in brain physiology, structure and function. The effects of alcohol are concentration-dependent and the patterns of excessive alcohol intake, such as binge drinking, are becoming predominant, especially in young drinkers. Alcohol binge drinking is defined as the pattern of consumption that increases the blood alcohol concentration above 80 mg/dL, which occurs after a consumption of about 56–70 g of pure alcohol in ≤2 h . At earl9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis, has an impact on brain development and function, and short term-memory deficits induced by THC have been well documented  in the hippocampus  for 5 weeks during adolescence, we explored the effects of OEA treatment on (1) spatial memory-like behavior, (2) mRNA expression of components of the neurotrophic –BDNF/NT3– system, (3) phosphorylation of intracellular protein kinases , and (4) cell proliferation, maturation, and survival in the dorsal hippocampus.Thus, here the main hypothesis under test was that OEA regulates BDNF signaling in a model of adolescent rats exposed to ethanol and THC. The homeostatic role for OEA has not been still explored in hippocampal functioning related to neurogenesis, BDNF-ERK signaling and memory that become notably altered by alcohol and partially affected by cannabinoid activation. Using a multi-disciplinary approach and a pharmacological administration of OEA  for 5 days/week in a rat model of binge-like consumption (1 day/week) of ethanol  together with acute administrations of ΔExperiments and procedures were conducted under strict adherence to the European Directive 2010/63/EU on the protection of animals used for scientific purposes and with Spanish regulations (RD 53/2013 and 178/2004). All efforts were made to minimize unnecessary suffering. All protocols were approved by the Ethics and Research Committee of Universidad de Málaga .ad libitum throughout the course of these studies.Animal studies were conducted on 5 to 10-weeks-old male Wistar rats  weighing ∼100 g at the beginning of the experiments. Rats were individually housed in clear plastic cages in a vivarium under standard controlled conditions: a 12-hour light/dark cycle (lights off at 8:00 pm.), ambient temperature (21 ± 2°C) and humidity (65 ± 5%); at the Animal House Centers of University of Málaga (Spain). Unless otherwise indicated, tap water and food (Purina chow) were available n = 80) were firstly habituated to the experimental conditions, including handling and injection procedures (holding and pseudo-injection) during one week before the experimentation in order to minimize stress effects (v/v) ethanol (3 g/kg) for five consecutive weeks  and/or the endogenous PPARα receptor agonist oleoylethanolamide . THC were dissolved in a solution containing 25% (v/v) ethanol in sterile 0.9% NaCl solution. OEA were dissolved in a vehicle containing 10% (v/v) Tween80 in sterile 0.9% NaCl solution. Both drugs were prepared just before each administration, and they were intraperitoneally (i.p.) injected in a final volume of 1 mL/kg of body weight. The optimal dose at which treatment would be more effective in behavior and neuroprotection, as described previously  was divided into five groups  and 5-iodo-2′-deoxyuridine  were dissolved at a concentration of 15 mg/mL in sterile 0.9% NaCl solution. To assess cell survival, all rats were treated with BrdU at a dose of 50 mg/kg (i.p.) for five consecutive days during the third week of experimentation . To asseThe effect of ethanol alone and combined with THC and/or OEA on short-term memory was evaluated using a Y-maze during the fifth week of experimentation. The maze has three interconnected closed arms, each one of them is 120° from the adjacent. The animal was placed into the middle of the maze and was allowed to freely explore the maze for 5 min. The movement of the animal inside the Y-maze was recorded by an overhead camera. The arms were thoroughly cleaned with a disinfectant solution with 70% ethanol and drying napkins to eliminate any residual odors of previous rats. Tests were performed 10 min after cleaning and 30 min after vehicle or OEA administrations. The number of arm entries were counted and acted as a marker of locomotor activity. The number of spontaneous alternations, as defined by entry into three different arms in sequence (triad), serves as a measure of short-term working memory. Percentage of spontaneous alternations was calculated from the number of triads and arm entries using the following equation: Y = number of triads /  × 100 .Previous to sacrifice, all animals were intraperitoneally anesthetized  in a room separate from the other experimental animals.n = 8/group) were sacrificed by decapitation and blood samples (n = 8/group) were briefly collected into tubes containing EDTA-2Na (1 mg/mL blood) and centrifuged . Plasma samples were then frozen and stored at −80°C for hormonal analysis. The brains were also collected, snap-frozen and stored at −80°C. These brains were then prepared on dry ice to obtain 1-mm-thick sections by using razor blades and a rat brain slicer matrix. The dorsal hippocampus was precisely removed from −2.16 to −4.20 mm of Bregma levels  were perfused transcardially with 4% formaldehyde in 0.1 M phosphate buffer (PB) and the brains were dissected out and kept in the same fixative solution overnight at 4°C. The brains were then cut into 30-μm-thick coronal sections by using a sliding microtome (Leica VT1000S) and divided in 12 parallel series. Sections were stored at 4°C in PB with 0.002% (w/v) sodium azide until they were used for immunostaining.A second batch of animals , following the manufacturer’s instructions. To perform the ELISA protocol in rat samples, we used 25 μL of plasma. A calibration curve and internal controls were included.Plasma levels of BDNF were determined using the enzyme-linked immunosorbent assay (ELISA) Invitrogen® method according to the manufacture’s instruction . RNA samples were isolated with RNeasy minElute cleanup-kit including digestion with DNase I column . After reverse transcript reaction from 1 μg of mRNA, RT-qPCR was performed in a CFX96TM Real-Time PCR Detection System  and the FAM dye label format for the TaqMan® Gene Expression Assays . Melting curve analysis was performed to ensure that only a single product was amplified. Ct values were normalized in relation to Actb levels.We performed RNA isolation, reverse transcription and RT-qPCR  as described previously  using spTM MP Imaging System (Bio-Rad). Western blots showed that each primary antibody detected a protein of the expected molecular size. The protein intensity was quantified with the image processing software ImageJ . The results were expressed as the protein/adaptin γ and phosphorylated/total protein ratios.Western blotting was performed as described previously . An appr2O2 in PB-saline.Free-floating coronal sections from −2.16 to –4.20 Bregma levels (hippocampus) of each parallel series were selected for each immunohistochemistry . Section5-bromo-2′-deoxyuridine and IdU-immunoreactive (+) cell nuclei that came into focus were manually counted using a standard optical microscope with the 40 × objective  coupled to the NIS-Elements Imaging Software 3.00 (Nikon). Brain structure analyzed consisted of approximately 8 coronal sections (from −2.16 to −4.20 mm Bregma levels), which resulted in one of every eight equidistant sections (one representative section for each 240 μm) according to the rostro-caudal extent. Estimations of the number of positive cells in the hippocampal neurogenic niche SGZ in both hemispheres were calculated per sections (30 μm deep), according to a rat brain atlas and cytoarchitectonic criteria . Quantifp-value less than 0.05 was considered statistically significant.Comparisons of data were carried out using IBM SPSS Statistical version 22 software  and GraphPad Prism version 6 software . Data are expressed as mean ± SEM for up to 8 determinations per experimental group . Plasma levels of BDNF were decreased in the rats exposed to ethanol compared to those of saline-vehicle ones (∗∗∗p < 0.001)  and THC  on the plasma levels of BDNF. OEA or THC alone partially reversed ethanol-induced BDNF reduction in plasma, because significant differences were still found compared to saline-vehicle ones . The combined administration of OEA and THC completely restored the decrease in the plasma levels of BDNF observed in rats exposed to ethanol (###p < 0.001) and THC/ethanol-exposed rats (&p < 0.05) (Significant changes in the circulating levels of BDNF were found (< 0.001) . Two-way < 0.05) . OEA at  < 0.05) .Bdnf, Ntf3, and TrkB , but not TrkC and Lngfr. The hippocampi of rats exposed to ethanol had higher mRNA levels of TrkB compared to those of saline-vehicle ones (∗∗p < 0.01) . A main effect of OEA on the mRNA levels of Ntf3 was found . Main effects of THC on the mRNA levels of Bdnf  and Ntf3  were also observed. OEA decreased hippocampal mRNA levels of Ntf3 in ethanol-exposed rats compared to those of ethanol-vehicle rats (#p < 0.05) and saline-vehicle rats (∗∗∗p < 0.001)  and saline-vehicle rats     . Two-way< 0.001) . THC dec< 0.001) . OEA in < 0.001) . The hip < 0.01) .F4,21 > 3.60; p < 0.026), total and phosphorylated ERK1 , and total ERK2  in the hippocampus. Specifically, the hippocampi of rats exposed to ethanol had higher total protein levels, but lower phosphorylated protein levels of ERK1 and ERK2 compared to those of saline-vehicle ones (∗p < 0.05)  and phosphorylated  protein levels of AKT were observed. Main effects of OEA on the total protein levels of ERK1  and ERK2 , and phosphorylated protein levels of ERK1  were found. THC increased total protein levels of AKT, ERK1 and ERK2 . OEA decreased total protein levels of AKT, ERK1, and ERK2 in ethanol-exposed rats compared to those of ethanol-vehicle rats   (&&p < 0.01) (#p < 0.05)  . Two-wayand ERK2 , but decand ERK2  in ethan < 0.01) . OEA inc < 0.01) . OEA adm < 0.01) . Contrar < 0.05) .Mki67, Dcx, Calb1, and Casp3 , but not Sox2 and Ncam. Specifically, the hippocampi of rats exposed to ethanol had lower mRNA levels of the cellular marker for proliferation Mki67 compared to those of saline-vehicle rats (∗∗p < 0.01) , Calb1 , and Casp3 . Main effects of OEA and THC on the mRNA levels of Dcx , Calb1  and Casp3  were also observed. The administration of OEA and THC alone decreased mRNA levels of Dcx, Calb1, and Casp3 in ethanol-exposed rats compared to those of saline-vehicle rats  and ethanol-vehicle rats  (Mk67 in ethanol-exposed rats (#p < 0.05), but this effect was erased when OEA was also administered  . Two-way< 0.001) . Specifinistered . No signobserved .F4,35 = 5.70; p < 0.0013), but not the number of IdU+ cells. Specifically, ethanol binges did not produce any effect on the number of BrdU+ cells  and the number of IdU+ cells (cell proliferation) in the SGZ of the dentate gyrus  and IdU+ cells  were observed. OEA alone did not modify the number of BrdU+ and IdU+ cells in the SGZ of ethanol-exposed rats. THC reduced the number of BrdU+ and IdU+ cells in ethanol-exposed rats compared to those of saline-vehicle rats (∗∗p < 0.01) and ethanol-vehicle rats (###p < 0.001)  . HoweverF4,35 = 3.10; p = 0.026), but not the number of arm entries , used as an index of activity. Specifically, ethanol binges did not produce any effect on spontaneous alternation or activity (arm entries) in the Y-maze  and the percentage of spontaneous alteration  were observed. THC affected both memory  and activity (arm entries) in ethanol-exposed rats (##p < 0.01)  in the Y-maze (e Y-maze . Two-way < 0.01) . This me < 0.01) .TrkB and the cellular factor for proliferation Mki67 in the hippocampus  and survival (Casp3 and BrdU+ cell population) in the dorsal hippocampus, (3) The repeated administration of OEA in the ethanol-exposed rats modulated the plasma levels of BDNF, and increased the hippocampal levels of phospho-AKT and phospho-ERK1, key signaling regulators of neurogenesis and cell survival (Ntf3), maturation  and pro-apoptotic (Casp3) factors in the hippocampus of ethanol-exposed rats, (5) Interestingly, the repeated administration of OEA in rats previously exposed to both ethanol and THC normalized the plasma levels of BDNF, enhanced the effects on the hippocampal mRNA levels of Bdnf and the neurotrophic receptors TrkB and TrkC, and sustained the phosphorylated levels of ERK1, and (6) These last effects were likely associated with a recovery of short-term spatial memory and newborn cell survival in the SGZ of the dentate gyrus , 48 h after acute administration of THC  and/or ethanol binge , on the mRNA levels of main components of the neurotrophic (BDNF and NT3) system, the phosphorylation of intracellular protein kinases , and the cell proliferation, maturation and survival in the dorsal hippocampus. The Y-Maze test was also tested as a behavioral assessment tool for short-term spatial memory. The main findings are as follows  Botte gyrus .Mki67, Sox2, Dcx, Ncam1, and Calb1 , a principal psychoactive component of cannabis that activates CB1 and CB2 receptors. Cannabis is the most widely used illicit drug, and the disruption of learning and memory are commonly reported consequences of cannabis use. In agreement with our results in ethanol-exposed rats, spatial memory impairment by THC has been demonstrated in adolescent animals  and healOleoylethanolamide is a satiety factor capable of controlling contextual memories associated with alcohol relapse . In addiDespite the association between OEA and BDNF has been barely studied, the chronic treatment with OEA markedly improves spatial cognitive deficits through enhancing neurogenesis and BDNF expression in the hippocampus after acute cerebral ischemic injury . SeveralBdnf and Ntf3 in the hippocampus. Concomitant OEA and THC did not restore neurotrophin expression but, synergistically, increased the mRNA levels of the receptor binding BDNF TrkB and decreased the mRNA levels of receptor binding NT-3 TrkC. Further studies are necessary to explain the apparent contradiction between the effects of THC and OEA on the hippocampal expression of BDNF and its receptor TrkB. Besides, synergistic reduction in Ntf3 and TrkC mRNA levels by THC and OEA suggest a relevant impact on NT-3 signaling system in a context of ethanol binges. This hypothesis is consistent with the intracellular signaling in the hippocampi of ethanol-exposed rats. The decreased levels of ERK1 phosphorylation induced by ethanol was specifically counteracted by OEA. We propose that this effect may be related to a sensitization of BDNF-TRKB signaling. Moreover, the increased levels of AKT phosphorylation induced by OEA agree with an activation of this signaling pathway  and neurogenic  factors in the hippocampus, a region widely related to synaptic plasticity of the neuron. Administration of OEA alone or in combination with THC in ethanol-exposed rats restored ethanol-related BDNF deficiency in plasma, increased the expression of the BDNF receptor TrkB and intracellular signaling (AKT and ERK1 phosphorylation), and sustained a reduced expression of maturation  and pro-apoptotic (Casp3) responses in the hippocampus. Finally, OEA may be related to a likely positive effect on newborn cell survival in the hippocampus and short-term spatial memory. Therefore, OEA and others putative PPARα activators are interesting targets to be further investigated to unveil novel therapeutic strategies approaching cognitive impairment and pathologies related to AUD.In conclusion, ethanol binge drinking during adolescence impairs circulating BDNF concentrations and ERK signaling in the hippocampus. THC associated with ethanol specifically induced a deleterious effect on both short-term memory and in a number of neurotrophic .FR and JS conceived and designed the study. DS-P, PR, FA, AV, and NG-M acquired the data. DS-P, PR, LR, FP, and AS analyzed and interpreted the data. DS-P and JS drafted the manuscript. FR and JS Reviewed and edited the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H  when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate , ethephon  and L-phenylalanine , added to previously selected variant of Murashige and Skoog medium  on the accumulation of harpagide and 8- O-cinnamoyl ester of harpagide–Harpagophytum procumbens (Burch.) DC. ex Meisn.–a medicinal plant currently listed in the European Pharmacopoeia [H. procumbens roots is limited. This is the reason for searching for other species containing harpagide and its derivatives or developing biotechnological methods to obtain these compounds [in vitro culture systems can offer alternative possibilities for the therapeutically important secondary metabolite source [Harpagoside . e source , 14). Nue source –20, haveO-acetyl-harpagide in M. melissophyllum subsp. melissophyllum agitated shoot cultures. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.The aim of the study was to investigate the influence of methyl jasmonate, ethephon and L-phenylalanine on the accumulation of harpagide and 8-Melittis melissophyllum L. (Lamiaceae) is a perennial plant inhabiting woody and shady areas in Central and Southern parts of Europe. The species contains a wide variety of chemical compounds that are responsible for its different medicinal properties used in the traditional medicine such as e.g. antispasmodic, digestive, activity against nervous anxiety, insomnia, eye inflammations, sore throat and cough [nd cough , 22.M. melissophyllum chemical compounds such as flavonoids [M. melissophyllum subsp. melissophyllum iridoidic fraction, isolating and identifying one new for the species compound allobetonicoside and the five previously known–harpagide , iridoiavonoids –29. Receavonoids  reassessarpagide , 8-O-acearpagide , melittoM. melissophyllum species includes three subspecies  P.W. Ball; subsp. carpatica (Klokov) P.W. Ball; subsp. melissophyllum L.), which differ in some morphological characteristics and occurrence area. M. melissophyllum subsp. melissophyllum grows, among others, in Poland. Italian researchers found that subsp. melissophyllum and subsp. albida differ in chemical composition of the essential oil [M. melissophyllum subsp. albida, apart from harpagide, 8-O-acetyl-harpagide, melittoside and allobetonicoside also contains such iridoids as virginioside and geniposidic acid, unknown for subsp. melissophyllum.tial oil  and iridtial oil . M. meliM. melissophyllum subsp. melissophyllum were obtained from Polish Botanical Gardens  in 2012 and 2015, respectively. The plant identity was verified by Dr Ewa Skrzypczak-Pietraszek, referring to available literature [in vitro cultures.Seeds and aerial parts of terature . Voucher3) 0.25 mg/L for multiplication. This PGRs variant was selected on the basis of our previous experience [-2s-1) for a number of 6-week subculture periods. Shoot cultures were used as an experimental material for subsequent biotechnological research.Shoot cultures were initiated from seeds in 2013. The seeds were surface sterilized according to the method described in , 34 and perience , 36. LitM. melissophyllum shoot cultures were cultivated on ten variants of MS medium , was carried out for the selected best production medium. Its conditions were: agitated shoot cultures on a rotary shaker (Altel), 140 rpm, the temperature and light conditions–the same as previously mentioned. All experiments were prepared in three independent series.The agitated shoot cultures for the supplementation experiment were initiated in 2016 by inoculating 3.0 g fresh mass of shoots in Erlenmeyer flasks (300 ml) containing 100 ml liquid MS medium (PGRs–the same as for the best growth and production medium previously selected). The flasks were placed on a rotary shaker (Altel) at 140 rpm, in the same temperature and light conditions as previously. Two weeks after inoculation, 100 ml of the same liquid medium was added. After another week cultures were supplemented with methyl jasmonate (Sigma-Aldrich), ethephon (Sigma-Aldrich) and L-phenylalanine (Merck) according to the scheme .® Millipore, 0.22 μm) before being administered to the medium. Variant A was considered as a control. Variants without MeJa, Eth or L-Phe were supplemented with sterile ethanol or water, respectively. Ethanol in the culture medium achieved a final concentration of 0.1% (v/v). The biomass and the medium (100 ml/flask) were harvested after 2 and 8 days, frozen and lyophilized. The dried biomass and medium were used to prepare extracts for the UPLC-MS/MS analysis. The experiments were performed with three independent samples for biological replicates.The stock solutions of L-Phe, Eth and MeJa had been prepared earlier. L-Phe and Eth were separately dissolved in water, MeJa was dissolved in 96% (v/v) ethanol and all three solutions were filter sterilized  and 1 g of each sample was extracted with boiling methanol . Extracts were combined (200 ml) and concentrated to dryness . Residues were dissolved in 10 ml of methanol . Residues of the lyophilized media were also dissolved in 10 ml of methanol . All methanol solution samples were filtered . All filtered samples were used for the UPLC-MS/MS analysis.The dried biomass from The developed UPLC method was chosen specifically for the examined compounds and it guaranteed that well shaped peaks were obtained. No interference that could have an influence on the obtained results was possible because the MRM method was used.−6), it was assumed that the calibration data fitted the quadratic model well. The correlation coefficient and determination coefficient  obtained for the model were over 0.99. The y-intercept of the quadratic equation was statistically significant (p < 0.004). The distribution of the residuals can be approximated well with a normal distribution as it is shown by p-value of the normality test (Shapiro-Wilk)–p > 0.63.Based on the regression analysis and Mandel’s fitting test . In all the deliberately varied chromatographic conditions , the examined compounds were adequately resolved, and the order of elution remained unchanged.The regression analysis results are presented in LC/MS-grade acetonitrile and LC/MS-grade methanol were from Sigma-Aldrich. Formic for LC/MS was from Sigma-Aldrich. HPLC grade water was obtained from HLP 5 (HYDROLAB Poland) apparatus and was filtered through the 0.2 μm filter before the use. Chloramphenicol  was from Sigma. Harpagide analytical standard  was from Sigma.Chloramphenicol internal standard was weighed to 10 mg in a volumetric flask using analytical balance. The volume was brought to 10 mL using methanol to make 1 mg/mL solution. 1 mL of this solution was subsequently diluted in a volumetric flask to 10 mL using methanol to make 100 μg/mL stock. The procedure was repeated to make 10 μg/mL stock solution. Harpagide standard was weighed to 1 mg in a volumetric flask using analytical balance. The volume was brought to 1 mL using methanol to make 1000 μg/mL stock solution. These solutions were stored at -10°C and used to make dilutions for calibration curves.A series of dilutions of the 1000 μg/mL harpagide standard solution was prepared by diluting 100 μL of the stock solution with water to make 1 mL, and afterwards diluting 500 μL of the obtained solution again with water to make 1 mL. The procedure was repeated several times to obtain finally a solution with the 0.186 μg/mL concentration of harpagide. 100 μL of the 10 μg/mL chloramphenicol internal standard solution was added to 500 μL of the dilutions of the 1000 μg/mL harpagide standard solution and diluted with water to 1 mL making calibration samples with concentration of chloramphenicol 1 μg/mL and concentration of the harpagide in range 12.5–0.098 μg/mL.100 μL or 50 μL of solution was taken from each sample, the 100 μL of 10 μg/mL chloramphenicol internal standard solution was added and brought to 1 mL of water making two dilutions of the stock solutions for each sample. Each dilution was analyzed in triplicate with the UPLC-MS/MS.O-acetyl-harpagide was created.In order to determine harpagide in the samples, the quantitative UPLC-MS/MS method was developed. Basing on this method the semi-quantitative method for determination of 8-® UPLC®  coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column, 2.1 × 100 mm, and 1.7 μm particle size. The column was maintained at 40°C, and eluted under following conditions: linear gradient elution from 100% to 90% of eluent A over 2 min, afterwards linear gradient elution from 90% to 0% of eluent A over 3 min and 100% of eluent B over 1 minute, at a flow rate of 0.3 mL/min. Eluent A: water/formic acid ; eluent B: acetonitrile/formic acid . 1 μL of each sample was injected in triplicate.The UPLC-MS/MS system consisted of a Waters ACQUITYO-acetyl-harpagide  at flow 0.28 mL/min. Optimized settings were as follows: source temperature 150°C, desolvation temperature 350°C, desolvation gas flow rate 600 L/h, capillary potential 3.00 kV, collision gas flow 0.1 mL/min. Cone potential and collision energy were individually optimized for each transition using a harpagide solution or a sample solution containing 8-arpagide . NitrogeAll analytical data was processed using MassLynx V4.1 software .The described method was validated to determine harpagide by the UPLC-MS/MS method according to ICH guidelines.Specificity. To demonstrate the specificity of the developed UPLC method the solution containing the investigated compounds was analyzed.System suitability. Possible interference between different compounds present in the analyzed samples was avoided by using the MRM method. Presence and identity of the compounds were verified by the presence of the peaks with appropriate retention times on both of the analyzed traces.Linearity. The linearity for the investigated compound was assessed by injecting eight separately prepared solutions covering the range of 0.098–12.5 μg/mL of harpagide. During the statistical analysis linear model and linearized nonlinear model (quadratic model) were analyzed:int/AUCint, where AUC–area under peak for the analyzed substance, cint−concentration of the internal standard in μg/mL, AUCint area under peak for the internal standard. For each compound peak only one trace was used in calculations.In the calculations response was used, defined as Response = AUC·c2 value and standard error of residuals of the calibration curve were calculated using the Statistica v.10 program. Next, to determine whether the residuals have normal distribution, the Shapiro-Wilk statistical test was used. Additionally the Mandel’s fitting test was performed to check the linearity of the calibration curve.The slope of regression line, y-intercept, standard deviations of the slope and intercept, a correlation coefficient, RLimit of detection (LOD) and limit of quantification (LOQ). Based on the standard error of residuals (SE) and the slope (a) of the calibration plots and following the definition of LOD and LOQ, i.e. LOD–the concentration estimated for the response equal to 10Se and LOQ–the concentration estimated for the response equal to 3.3Se, the LOD and LOQ for examined compounds were estimated.Precision. The repeatability of the method was checked by a sixfold analysis of the 6.25 μg/mL concentration level of harpagide solution. The same protocol was followed for three subsequent different days to study the intermediate precision of the proposed method. The RSD (%) of the peak area of harpagide was calculated. Statistical significance of interday differences was tested with ANOVA.Robustness. To demonstrate the robustness of the method, small changes of flow rate, content of acetonitrile and column temperature were deliberately made in the relation to the optimal values. The mobile phase flow rate was 0.30 mL/min; to study the effect of the flow rate on resolution, the flow rate was changed to 0.27 and 0.33 mL/min. The effect of the column temperature was studied at 36°C and 44°C (instead of 40°C), and the mobile phase composition was changed by +5% from the initial composition.The investigation was prepared according to the rules of the designed experiment . Four coO-acetyl-harpagide , a total concentration of harpagide and 8-O-acetyl-harpagide  and a total yield of harpagide and 8-O-acetyl-harpagide per flask .Five quantitative outputs were selected: a dry weight , a concentration of harpagide , a concentration of 8-4 experimental design [The two-level full factorial 2l design  with 16 The factor settings are presented as coded values in Yates’s manner (-/+) The fixed effects model was used to analyze the obtained data . InitialThe initial model was a fixed effects model with interactions up to and including the fourth order. The analysis of effects, combined with the estimation of a pure error on the basis of replications, allowed to determine statistically significant terms of the model  visuallyAll the computations were performed using Minitab and Statistica software.M. melissophyllum shoot cultures prior to elicitation and precursor supplementation. Ten variants of MS medium supplemented with different PGRs combinations were used , ethephon  and L-phenylalanine , in different combinations , on the content of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures  .The differences, however, were not statistically significant. For variant D, the corresponding values were similar to variant A. The average dry weight of the plant material harvested after 8 days showed greater variability. It was the highest for variant A representing the control. The addition of L-Phe, MeJa and Eth (in various combinations) caused a slowdown in the growth of the cultures. A particularly strong reduction in weight could be observed for the variants containing L-Phe in their composition . The lowest average weight of the biomass was found for variant H (L-Phe together with MeJa and Et), which was about 40% lower than the average weight of the biomass harvested from the control culture (variant A). Moreover, for variants E, F, G and H, slight partial browning of the plant material was observed. This may indicate some damage to plant tissues and consequently lead to the release of iridoids into the medium. The HPLC analysis of the media collected from the cultures confirmed this supposition. The presence of the metabolites in the medium was found for those particular variants  harvested eight days after the time of modification.It was found that each modification of the medium, consisting in the addition of L-Phe, MeJa and Eth in various one-, two- and three-component combinations (variants B-H), increased the accumulation of harpagide (Hp) in the biomass, in comparison with the control culture (variant A) .In the plant material harvested two days after the modification of the medium, the highest Hp content was observed for variant F (L-Phe and Eth), which was 247.3 mg/100 g DW, and was approx. 10 times higher than in the control. The addition of L-Phe in conjunction with MeJa (variant E) or L-Phe in conjunction with both elicitors (variant H) was less advantageous than variant F, and caused similar increases in accumulation– 136.7 and 137.0 mg/100 g DW respectively (5.7-fold increase). Individual additions of only L-Phe (variant B) or only Eth (variant D) resulted in only a slight increase in accumulation– 2-fold and 1.4-fold, respectively.Comparing the average Hp content in the biomass harvested 8 days and 2 days after the modification of the medium, it can be stated that only for variants B, D and E there was an increase in the accumulation of the metabolite, and for the other variants–a decrease in content. For variants E, F, G and H, one must, however, take into consideration the fact that a portion of Hp had been released into the medium. For variants E and G, the greater part of Hp was located in the biomass than in the medium, whereas for variants F and H the amount of the compound was approx. 3 times greater in the medium than in the plant material. The highest average Hp content in the biomass was found for variant E (192 mg/100 g DW). Considering the total amount of the compound , the highest accumulation of it (far exceeding the content in the biomass harvested 2 days after modification) was observed for variants F and H– 619 and 428 mg/100 g DW respectively .O-acetyl-harpagide (AcHp) content in the control cultures (variant A) was approx. twice as high as the average Hp content in analogous cultures , reaching the maximum average content of 138 mg/100 g DW for variant E. After another 6 days (i.e. 8 days after the modification of the media), the situation changed, and an increase in AcHp in the biomass was found for other variants–B, F and H, where it reached the maximum of 114.2 and 110.7 mg/100 g DW (variants B and F respectively). However, considering the total AcHp content (in both the biomass and medium), the highest average content of 255.4 mg/100 g DW was found for variant H.AcHp is an ester derivative of Hp. They share the same biosynthetic pathway, and therefore the accumulation of the two compounds in the cultivated cultures should be considered jointly .-1 day-1, respectively), 3.9 and 3.6-fold higher, respectively, comparing with the control culture–variant A  was found for variants G, E and F, where it reached the maximum of 293.3 mg/100 g DW . The higariant A .-1 day-1, respectively), 6.3 and 4.9-fold higher, respectively, comparing with the control culture–variant A  was found for variants F and H– 776.7 and 683.4 mg/100 g DW respectively. In comparison with the control culture there was, respectively, a 10-fold and 8.8-fold increase in the combined amount of the two metabolites . The higariant A .M. melissophyllum from both Botanical Gardens did not differ significantly in statistical terms. The Hp content in the shoots of the flowerless (not flowering) soil-grown plant of M. melissophyllum was 41.3 mg/100 g DW, and the AcHp content– 37.9 mg/100 g DW. These amounts were, respectively, approx. 72% higher and approx. 27.8% lower than in the biomass harvested from the control culture (variant A). This is quite different in the case of Rehmannia elata N.E. Brown ex Prain, a plant that also contains Hp. Shoot cultures of this species accumulate from 13 to 25 times more Hp than the shoots of the soil-grown plant [M. melissophyllum contain only small amounts of Hp and AcHp, 2.7 mg/100 g DW and 2.1 mg/100 g DW respectively, many times lower than the shoots . A similar relationship was observed for soil-grown plants of Scrophularia nodosa L. [Scrophularia yoshimurae Yamazaki accumulates Hpd in both the roots and the aboveground parts [The content of iridoids in aerial parts of wn plant . The steodosa L. . The leand parts , 24).The medium variants that are optimal for the accumulation of the tested metabolites, in the case of harvesting the biomass 8 days after the modification of the medium, are variants F and H. The amounts of Hp and AcHp in the biomass from in vitro cultures are then many times higher than in the leaves of the soil-grown plant: about 15 times and about 4 times respectively for variant F, and about 10 times and almost 7 times respectively for variant H.Taking into consideration the composition of variants F and H, it can be concluded that the crucial addition is that of L-Phe together with Eth (variant F). The use of L-Phe with two elicitors (variant H) more considerably increases the accumulation of AcHp than Hp, whereas the use of L-Phe only in conjunction with MeJa (variant E) is not optimal for either of the two metabolites.in vitro the accumulation of metabolites [Psoralea carylifolia increased 2.8-fold with 1 μM jasmonic acid (JA) treatment after second week and 7.3-fold with 10 μM JA elicitation after 10th week compared to untreated control [Andrographis paniculata. Andrographolide production after the first week increased 3.5-fold with 1 μM JA and 10.8-fold with 25 μM JA compared to control [Andrographis paniculate increased 2.6-fold with 1 μM JA after fifth week but JA treatment at 25 μM promoted only 3.3-fold enhancement in metabolite content after eighth week compared to control [The use of elicitors is a known method of enhancing in cultures abolites  from varabolites –46. It i control . JA was  control .Panax ginseng C.A. Meyer.Sometimes, a better result is obtained through the simultaneous use of two elicitors. According to Bae, Choi , the comIt is puzzling that the addition of L-Phe increases the accumulation of Hp and AcHp to a particularly high extent when it is added together with Eth, or Eth and MeJa. L-Phe is an aromatic amino acid biosynthesized via the shikimic acid pathway. It is a precursor of many groups of chemical compounds, in particular derivatives of phenylpropane. The metabolic pathway leading to the biosynthesis of harpagide is still not fully understood –52, but in vitro. One of such studies describes the influence of growth regulators on the accumulation of several metabolites (incl. Hp) in shoot cultures of Rehmannia elata N.E. Brown ex Prain [M. melissophyllum shoot cultures with the results presented above, it can be noticed that the Hp content in the biomass harvested from the optimal variant (F) is approx. 6 times higher than that for R. elata cultures.There are not many publications concerning research on increasing the accumulation of Hp in cultures ex Prain . The medex Prain  containiRehmannia glutinosa Libosch. The highest Hp content of 0.136 mg/g DW was obtained 72 hours after the addition of 150 μM MeJa. This content was 7.5 times higher than the one for the control culture, without the addition of elicitors. The simultaneous addition of MeJa and SA did not produce such a beneficial effect. The maximum Hp content in M. melissophyllum shoot cultures is up to 45.5 times higher than in the hairy root cultures of R. glutinosa. The authors [Exacum affine [In another study , the inv authors  found thm affine .Harpagophytum procumbens plants is Hpd. Apart from this compound, other iridoid glycosides, incl. Hp, are present in smaller amounts. According to the European Pharmacopoeia [H. procumbens, a study examined the influence of the method of cultivating the culture (in shake flasks and in a 3-litre bubble column bioreactor) on the accumulation of Hpd and Hp in the biomass [M. melissophyllum in a bioreactor in the future. The maximum Hp content in the hairy roots of H. procumbens was only approx. 4.5 μg/g DW. In other experiments with hairy root cultures of this species [in vitro cultures were also considerably lower than in the raw material from the soil-grown plant, and were as follows: 0.32 mg/g DW  and 0.6 DW (Hpd ). Those  species , the totocumbens  there wet at all . The autt at all  suggest Statistical analysis confirms previously mentioned observations.The analysis of the results (48 records) started from the fixed-effects model with interactions up to fourth order. Pareto plot of standardized effects  revealedThe insignificant model terms were removed and the analysis was performed again for HTime, L-Phe and their interaction. Pareto plot of standardized effects  confirme2 = 0.43. Such value of R2 suggests that 57% of unexplained variance is caused by other factors: controlled, but ignored (because of their insignificance) MeJa and Eth, and also other environmental and material factors not considered here.ANOVA analysis  reportedThe model residuals  followednd day and the concentration of L Phe 2.4 g/L. The dry weight predicted for such settings was 3.05 with 95% confidence interval .The final model predicted that maximum dry weight was obtained for HTime = -1 and L-Phe = +1 , which mThe first-step analysis of dataset reported strong heteroscedasticity of data and the Box-Cox procedure was performed . After dth-order model. It reported insignificance for the term of the three-way interaction HTime x L-Phe x Eth .The maximum harpagide outcome was predicted undoubtedly for HTime = +1, L-Phe = +1, MeJa = -1 and Eth = +1 , two two-way interactions  and one three-way interaction (L-Phe x MeJa x Eth)  .After removing these terms, the ANOVA analysis was performed again and it reported a well-fitted model with residuals  followinO-acetyl-harpagide was, because of the reciprocal transformation, located at the minimum of transformed outcome associated with the first homogenous group .Next, Tukey’s HSD test was performed and it revealed 5 homogeneous groups inside the dataset . The maxus group . The optth day, the concentration of L-Phe 2.4 g/L and the supplementation with the ethephon 50 μM. The maximum 8-O-acetyl-harpagide outcome predicted for all settings on +1 (with MeJa supplementation) was 305 mg/100 g DW with 95% confidence interval .It means, in the real world, the harvesting time on the 8O-acetyl-harpagide outcome.The first-step analysis of the dataset reported strong heteroscedasticity of the data and the Box-Cox procedure was performed . After dth-order model. It reported insignificance for two terms: two-way interaction (L-Phe x Eth) and one three-way interaction (HTime x L-Phe x Eth)  .After removing these terms, the ANOVA analysis was performed again and it reported a well-fitted model with residuals  followinO-acetyl-harpagide was, because of the reciprocal transformation, located at the minimum of transformed outcome associated with the first homogenous group . It means, in the real world, the harvesting time on the 8th day, the concentration of L-Phe 2.4 g/L and the supplementation with the ethephon 50 μM. The maximum of the total of harpagide and 8-O-acetyl-harpagide outcome predicted for all settings on +1 (with MeJa supplementation) was 761 mg/100 g DW with 95% confidence interval .The optimum conditions for the maximum of 8-O-acetyl-harpagide outcome per flask.The first-step analysis of the dataset reported strong heteroscedasticity of the data and the Box-Cox procedure was performed . After dth-order model. It reported insignificance for two terms: one main effect, four two-way interaction and three three-way interaction  while three experimentally observed values for this treatment were 12.41, 19.09, 20.12 mg/flask.It means that six different treatments constitute possible optimum settings statistically proved however the greatest prediction of the total productivity was HTime = +1, L-Phe = +1, Eth = +1 with the lack of MeJa. It means, in real world, the harvesting time on the 8O-acetyl-harpagide in M. melissophyllum shoot cultures has been reported. We can conclude that elicitation with MeJa and Eth, and medium supplementation with L-Phe significantly increased the accumulation of harpagide and 8-O-acetyl-harpagide in M. melissophyllum agitated shoot cultures. The highest amounts of iridoids were detected on the 8th day after medium supplementation: harpagide 0.62% DW , 8-O-acetyl-harpagide 0.26% DW  and the total amount of the two iridoids 0.78% DW (variant F). The results indicate that M. melissophyllum agitated shoot cultures might be considered as a potential biotechnological source of harpagide and 8-O-acetyl-harpagide.To the best of our knowledge, it is the first time that the analysis of harpagide and 8-S1 Dataset(XLSX)Click here for additional data file.


This study aimed to isolate some of the currently circulating infectious bronchitis virus (IBV) strains from some broiler chicken farms in Al-Hasa and to do some molecular characteristics of these strains.We collected 300 tissue specimens, including the trachea, bronchi, lungs, and kidneys from some four commercial chicken farms showing respiratory manifestations. We tested these tissue specimens by the real-time polymerase chain reaction (RT-PCR) and gel-based PCR. We selected some PCR positive samples for isolation in the embryonated chicken eggs (ECE). We sequenced some PCR-positive samples and conducted phylogenetic analysis based on the obtained sequences.Our molecular surveillance revealed that 31.6% of the tested specimens were IBV positive by PCR. We selected some positive specimens showing low Ct values by the qRT-PCR for virus isolation by the ECE. The infected eggs showed hemorrhage, dwarfing, and death in some cases after three passages in the ECE. We sequenced some of the positive PCR specimens and used the obtained sequences to draw the phylogenetic tree based on the partial IBV-ORF-1a, N, and S1 gene sequences. The phylogenetic trees based on the IBV-N and S1 gene sequences showed that the circulating IBV strains in Al-Hasa during 2016 was showing a high degree of identity to some strains from Taiwan and Italy. Meanwhile, the grouping of these strains based on the IBV-S1 sequences revealed that the currently circulating IBV strains in Al-Hasa belonged to Gr.I.7 along with strains from Taiwan.Our results confirmed the continuous circulation of the IBV among the chicken population in Al-Hasa despite the intensive application of vaccines against this virus. Nidovirales, family Coronaviridae, and genus Gammacoronavirus [Escherichia coli and Mycoplasma gallisepticum) always exaggerate the viral pathogenesis and auscultate the mortality rates among the infected chicken population [Infectious bronchitis virus (IBV) is a highly contagious respiratory viral disease of chickens of all ages. The IBV belongs to the order onavirus ,2. The Ionavirus . This stonavirus . Based oonavirus . The IBVonavirus . The IBVonavirus . Secondapulation . The IBVpulation .Efforts to control spreading of the IBV infections through vaccination resulted in some variable outcomes. Many IBV strains and serotypes have emerged since its discovery >80 years ago; meanwhile, the misuse of the IBV vaccines complicates the evolution and emergence of new IBV strains ,10. ManyThe IBV infection in chicken was first reported in Saudi Arabia in 1984 in field samples by the real-time polymerase chain reaction (RT-PCR) using N primers . AnotherSeveral IBV variants and genotypes are currently circulating in the Middle East, Asia, and North Africa such as Iraq, Egypt, Libya, Iran, and Jordan . FurtherThis study aimed for isolation and molecular characterization of the IBV circulating strains of the IBV in Al-Hasa in the eastern region of Saudi Arabia. This region is one of the major hubs for intensive poultry production in Saudi Arabia.http://www.kfsh.med.sa/KFSH_WebSite/usersuploadedfiles%5CNCBE%20Regulations%20ENGLISH.pdf). The Animal Ethics Committee of the King Faisal’s University approved this protocol.We conducted this study according to the King Faisal’s University Animal Ethics protocols and the National Committee of Bio-Ethics, King Abdul-Aziz City of Science and Technology, Royal Decree No. M/59  and proved to be IBV-negative antibodies by ELISA. The inoculum was prepared as follows; IBV-suspected tissue suspensions were centrifuged for 10 min at 5000 rpm. Antibiotic mixture was added (penicillin and streptomycin) to each tissue suspension. We used 100 µl per each tissue suspension to inoculate through allantoic route into five embryonated eggs . The inoThe total viral RNAs were extracted from tissue using the QIAamp Viral RNA Mini Kit  was processed in parallel to each batch of specimens as described above.The RT-PCR was performed using the IBV RT commercial kits  . The reactions were conducted as per the kit’s instructions with some minor modifications. Briefly, each reaction consisted of 2×RT-PCR master mix  in a total reaction volume of 20 µl. Meanwhile, we used 5 µl of nuclease-free water as negative control. Furthermore, a positive control reaction was carried out in parallel to each run. Duplicate reactions were done per each specimen. The cycling parameters were 55°C for 10 min, then 95°C for 8 min, followed by 50 cycles at 95°C for 10 s, and 60°C for 1 min.We designed some primers to amplify the partial IBV-N gene. We designed the IBV-N primers based on the IBV-N (accession no: KT762154). The nucleotide sequences of the used IBV-N gene are IBV-NF (5’-CGCTGGAGAATTTCCTCTTG-3’) and the IBV-NR (5’-CTAGTCCCTAGCAGCCATGC-3’). Meanwhile, we used the following primers, IBV-S1F (5’-ACCGGCTGATGGATGGCAT-3’) and the IBV-S1 (5’-TTGCTTACAACCACCCTGTAG-3’), to amplify the partial IBV-S1 gene.The RT-PCR reactions were conducted in a 25 µl reaction volumes using the one-step RT-PCR kit . The reactions were conducted as per the manufacturer’s instructions. The reaction mixture consists of 10 µl of 5×reaction buffer, 2 µl of each primer, 2 µl of enzyme mixture, 2 µl of dNTP mixture (400 µM of each dNTP), 3 µl of the RNA template, and 4 µl of RNase-free water. Reverse transcription was started at 50°C for 30 min; initial Taq polymerase activation was performed at 95°C for 15 min, along with 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and then extensions at 70°C for 1 and 10 min.For visualization of the amplified PCR products, electrophoresis of 10 µl of each PCR products on 1% agarose gel was performed. This was done under the ultraviolet light and photographed by the Bio-Rad gel documentation system. 10 µl of per each PCR product was mixed with 3 µl of the loading buffer . A 100-bp, ready-to-load DNA-marker ladder was loaded in a separate, parallel lane.We processed a confirmed PCR-ECE-P3-IBV isolate for sequencing by the Illumina next-generation sequencing approach. We used this approach to decode the partial-length genome sequences of this IBV isolate as previously described .We constructed the phylogenetic tree based on the obtained partial IBV-ORF1-a, IBV-N, and S1 gene sequences. We constructed the tree using the multiple alignments of these sequences with other IBV sequences retrieved from the GenBank done using the Mega-7 package, and phylogenetic analysis was done by the maximum likelihood method using the best-fit model determination and at least 500 bootstrap replicates as previously described . MeanwhiWe applied the non-probability sampling strategy for our specimen collection with incidental assignment approach as previously described .The IBV infection in chickens produces a wide range of clinical syndromes. Some birds showed respiratory manifestation in terms of coughing, sneezing, and nasal discharges. Necropsy examination of some IBV-infected birds revealed serous, catarrhal, or caseous exudate in the tracheal, nasal passage, sinus, and bronchi congestion . The edeWe conducted molecular surveillance to check the prevalence of IBV across the Al-Hasa from October 2015 to April 2016. We investigated four IBV outbreaks across the Al-Hasa province by the RT-PCR . We testWe already tested different tissue specimens, including the trachea, lungs, and kidneys, from some suspected IBV-infected birds. Our results are showing that 11 tracheal tissue specimens of 20 were positive, while 6 lung specimens of 20 were positive; meanwhile, only two specimens from the kidneys were positive .We processed 15 specimens  after pooling from three IBV outbreaks. We did the propagation for three passages. The IBV isolation was successful only in case of two specimens. The first specimen was collected from an RT-PCR-IBV-positive chicken trachea collected from farm no. 7. The second specimen was collected from positive IBV-RT-PCR lungs collected from chicken farm no. 5 area . HoweverWe used three tissue suspensions  from three specimens representing three different IBV outbreaks in some chicken farms from Al-Hasa region. We followed up these three inoculums on the ECE for three subsequent passages. The IBV-infected tracheal specimen caused death and induced some pathological changes in two embryos from the inoculated embryos in the three passages. However, one of them showed no pathological changes or death at the end of the experiment. Meanwhile, the IBV-infected lung tissue specimens showed that only one embryo was affected per each passage.We used commercial available RT-PCR kits to confirm the identity of some suspected IBV-infected chicken tissues, as well as some fluids from the embryonated egg passages. We carried out the RT-PCR amplification for three representative specimens from the ECE passages P-1-3. Meanwhile, we already included six original specimens from the IBV-suspected outbreaks from different farms, including some pooled tissue specimens . Furthermore, we already included the IBV vaccine as a positive control of the reaction in addition to the kits positive control in the reactions. In addition to a negative control used a non-DNA template. Our results show that 6/9 (66.67%) of the three tested specimens representing embryonated eggs passages 1-3 were positive. Three specimens from selected IBV specimens were positive, as shown in Based on the reported IBV-ORF1a sequences, these strains were closely related to IBV-L1148 strains . The phyBoth the pairwise distance  and the The geographical distribution of the suspected outbreaks represents different regions across the Al-Hasa. Some chicken farms were selected around the Al-Hasa region as targets for our study. Some of these farms were reporting IBV outbreaks based on the obvious clinical signs and post-mortem lesions, while birds in some others were apparently healthy. The clinical examination of the affected birds showed depression, ruffled feathers, loss of the body weight, respiratory rales, nasal discharge, and lachrymal discharge (data not shown). Necropsy examination of some affected birds revealed congested trachea, caseous and bloody plugs at the tracheal bifurcations , congestWe conducted an IBV molecular surveillance among some selected chicken farms in Al-Hasa region by the RT-PCR technique using the conserved IBV-N primers. We tested 100 specimens from layers, and broiler non-vaccinated chicken’s flocks represented four suspected IBV outbreaks across the Al-Hasa. These tissue specimens were collected from various organs of the affected birds such as the trachea, lungs, and kidneys. We did pooling five organs per each tube considered as one specimen. We found that 19/60 specimens (31.67%) were positive . Our resWe used the generated sequences of three independent genes  in the IBV genome to do the phylogenetic analysis of the circulating strains in Al-Hasa region during 2016. We found great overlapping and consistency of the phylogenetic analysis based on the IBV-S1 and N genes. The reported sequences were closely related to other IBV strains reported in Taiwan, Italy, and China Figures--5. This Our results confirmed recent reports about the circulation of different IBV strains in the kingdom and the eastern region in specific ,29. ThisIBV continues to pose a great risk to the poultry industry in the Al-Hasa, Saudi Arabia. There is continuous circulation of the IBV between the broiler and layer chicken in this area. The currently circulating strains of the IBV belong to the Gr.I-17 along with other genotypes from Taiwan, China, and Italy.MGH designed and performed the experiments, oversee the entire work, and performed data analysis and interpretation. MuAA conducted the fieldwork, performed some laboratory experiments, and drafted the manuscript. MoAA helped in the data analysis and reading of the manuscript. All authors read and approved the final version of the manuscript.


Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta. Myofibrroblasts . In 2D croblasts , 10. Thiroblasts . The preroblasts , 13.in vitro contractile properties of the human placenta [2+ concentration; 3°) they conform to the Frank-Starling mechanism [Earlier studies have assessed the placenta . This noplacenta , which aechanism , 16: 4°)echanism . Such a echanism  and, in echanism . The molechanism , 21. NMMechanism , 22, hasechanism  and MSC-The solid 3D collagen scaffold were functionalized with the linear Arg-Gly-Asp (RGD) before MSC-seeding. The covalent binding of the linear RGD motif to same solid collagen scaffolds has been shown to improve the contractility of rat cardiomyocytes seeded in such structure . In thisThis study was approved by the local ethics committee of Geneva University Hospital, named « Commission Cantonale Ethique de la Recherche Scientifique de Genève » (CCER). The human femoral heads were collected during surgical interventions for hip replacements, according to the CCER, and after patients were informed and gave their written consent. MSC extraction, amplification and MSC seeding in 3D-solid collagen scaffolds have been previously described in detail . Thus, B™ Ultrafoam collagen  with the linear RGD motive of fibronectin has been reported [Scaffolds are a solid highly porous collagen structures obtained by physical reticulation by dehydrothermal (DHT) polymerisation of bovine dermal collagen type I and type III. Scaffolds are commercially available for clinical implantation as hemostatic sponge. The 3D structure of collagen RGD scaffolds was obtained by electron microscopy showing a classical ultrastructure of sponge . The funreported . Structu-1. cm-1). The level of SPDP-modification was monitored by the release of pyridine-2-thione in the medium. Moles of SPDP per mol of protein (GRGDS) is equal to the absorbance change at 343nm divided by 8080 and multiplied by the molecular weigth of GRGDS and divided by mg/ml of GRGDS. With this protocol for 10 mg of collagen, we achieved a level of peptide functionalization of 70±4%; n = 7). MSCs were used after 4 passages and 105 MSCs were seeded in each scaffold. The time during which MSCs were cultured in scaffolds was 3 to 4 weeks. Some preliminary experiments were done after 8 days of culture and this led to the same level of MSC-seeded scaffold contractility.The full linear RGD peptide of fibronectin, i.e., glycine-arginine- glycine-aspartic acid-serine peptide  was used. Briefly, for functionalization, the RGD motif needs to be bound to the scaffold. The use of a spacer arm between 30–40 A is optimal for the RGD presentation and favorable for integrin-receptor clustering and activation , 25. The2) from the weight/Lo ratio. Scaffolds were chemically stimulated by KCl (0.05M). The electromagnetic force-length transducer has been previously described [BM-derived MSC-laden scaffolds, both with and without the RGD motif, were used to test their contractile properties. Each scaffold was mounted in an experimental chamber containing a Krebs-Henseleit solution, bubbled with 95% O2–5% CO2 to insure a pH of 7.4. Experiments were carried out at room temperature, and at resting length (Lo), i.e., with a resting tone (RT) that induced neither spontaneous shortening nor spontaneous lengthening of the scaffold. At the end of the experiment, the cross-sectional area (CSA) of the scaffold was calculated  imposed on the scaffold and σ = F/CSA  was the force F (in N) per CSA (in m = F/CSA . The str = F/CSA . The str = F/CSA .Scaffolds were preloaded at RT. KCl (0.05M) was then introduced in the experimental chamber. This induced an immediate isotonic shortening of the scaffold . A plateThe Frank-Starling mechanism , 16 was −2) was measured from fully isometric conditions. Vmax was the maximum shortening velocity at zero load. The G curvature of the T-V relationship was TT / a = Vmax / b [The A.V. Hill hyperbolic tension-velocity (T-V) relationship  was deteVmax / b . V was t1 and g2 were the maximum values of the rate constants for CB detachment; w was the maximum mechanical work of a unitary CB (w / e = 0.75) and e was the free energy required to split one ATP molecule. The standard free energy ΔG°’ATP was—60 kJ /mol. The value used for e was 10 −19 J [1 and g1 corresponded to a tilt from 0 to h and g2 corresponded to a tilt > h; Φ = (f1 + g1) h / 2 = b; N was the number of cycling CBs per mm2 at peak isometric tension. The molecular step size h represented the translocation distance of the actin filament per ATP hydrolysis, produced by the tilt of the myosin head. Parameter ℓ represented the distance between two successive actin sites with which any myosin site can combine. According to the A. Huxley conditions (ℓ >>h), the h and ℓ values were h = 10 nm and ℓ = 28.6 nm, which is close to the semi-helicoidal turn of the actin filament [1, g1, and g2 were obtained from the following equations [−1) and the Avogadro number. The maximum NMMIIA-ATPase activity was the product of the catalytic constant  and NMMIIA content. The rate of mechanical work (WM) was equal to the product PHux x V [M and EHux. Maximum efficiency (Eff.max) was the maximum value of CB efficiency.The A. Huxley formalism  enables 10 −19 J . The tilfilament , 30. Valquations :G=f1/g1PHux x V . At any Data were expressed as means ± standard deviation (SD). Student’s unpaired t-test was used for comparisons of parameters between collagen scaffolds seeded with MSCs and functionalized or not, with RGD. On Figures, *: p < 0.05; * *: p < 0.01; * * *: p < 0.001. NS: non significant. A p value < 0.05 was considered statistically significant. Linear regressions were performed by means of the least squares method.3, the maximum ATPase activity of the NMMIIA, the force of a single actin-NMMIIA CB , and their passive mechanical properties (subsection 2: Passive parameter: The Young modulus of scaffolds seeded with MSCs)  were preMMIIA CB , the kinMMIIA CB . The RDGSixteen control scaffolds and ten RGD-decorated scaffolds were analyzed. The basic physical properties of the scaffolds laden with MSCs, i.e. the properties observed before the application of the KCl stimulus , are presented in The Young modulus was established for each type of scaffolds laden with MSC . It averThe maximum rate of shortening at zero-load (Vmax) and total isometric tension (TT) was significantly greater in RGD scaffolds than in the control scaffolds (p = 0.001) Figs .With 0.05 M KCl, the gradual stretching of a scaffold by increments of 0.1 mm increased the isometric tension linearly . The sloThe number of nanomoles of NMMIIA-CB per gram of tissue was 5 times greater (p = 0.0001) , while t3, the maximum ATPaseactivityoftheNMMIIA (thekineticsofattachment (f1) anddetachment (g1 and g2) of the actin-NMMIIA CB and the catalytic constant (kcat) , amplified in 3D cultures and complemented with HPL [In this study, we devised an artificial contractile system comprising human bone marrow cells (MSCs) seeded into 3D-collagen scaffolds with and without the RGD motif. These 3D-collagen scaffolds exhibited similar contractile properties to those of the human placental tissue , 33, 34.eNMMIIA , thekint (kcat) . The RGDachment f anddetawith HPL , 19 and with HPL . MoreoveIn the present study, experiments were performed within the same period and with the same biological samples used in our previous study . Thus, winitiallength by increasing the degree of overlapping of the number of active CBs on NMMIIA filaments along α-SMA filaments [Surprisingly, we observed that under KCL stimulation, MSC-collagen scaffolds exhibited the Frank-Starling mechanism, i.e. the active isometric tension increased with increases in initial length , 16, 35.ilaments . The lowAmong the mechanical properties of the MSC-laden scaffolds, the second surprise was the observation of the hyperbolic tension-velocity relationship , 37. ThireinforcestheideathattheFrank-Starlingmechanismis underpinnedbythe overlap of NMMIIA and α-SMA filaments andthatthepreloaddeterminesthenumberof NMMIIA-CBsthatareinteractivewith overlapping α-SMA filaments. Conversely, the unitary force of each CB (We showed that the functionalization of the solid collagen scaffold with the RGD motif in the presence of MSCs increased the contractile properties of preparations. All scaffolds containing MSCs decorated with the RGD motif exhibited major changes compared with control collagen scaffolds: maximum shortening velocity and total isometric tension increased in the presence of the RGD motif . The inc each CB  and the  each CB  were not each CB  without  each CB .The Young modulus has previously been shown to be significantly higher in scaffolds seeded with MSCs than in those without MSCs . While tRGD has been first described as a sequence within fibronectin that mediates cell attachment. The linear RGD is the main adhesion molecule for integrin receptors αvβ5, αvβ3 and α5β1. Interactions of integrin receptors with small adhesion peptides present on proteins of the ECM such as the collagen or fibronectin are known to mediates both cell-substratum and cell-cell interactions . The funWe designed an artificial functional contractile tissue comprising human MSCs seeded in collagen scaffolds whose contractility could be improved by coupling the RGD peptide to the collagen prior to scaffold-cell seeding. The fundamental mechanical properties observed in all striated and smooth muscles were also observed in MSC-laden scaffolds, namely the Frank-Starling phenomenon and the A.V. Hill T-V hyperbolic relationship. The Frank-Starling phenomenon indicated that there was a relatively high level of ultrastructural organization of actin and NMMIIA molecules within the cells residing in collagen scaffolds, probably allowing actin filaments to slide along non-muscle myosin IIA filaments. Moreover, the chemical binding of the RGD peptide to the collagen scaffolds modified certain fundamental molecular properties of NMMIIA-CBs within the myofibroblasts. This led to an increase in contractility characterized by an increase in maximum shortening velocity and total tension. At the molecular level, the RGD motif increased the kinetics of detachment of NMMIIA-CBs as well as the molar NMMIIA concentration and the maximum NMMIIA-ATPase activity without altering the unitary force of NMMIIA-CBs and their maximum efficiency. Thus the artificial tissue we designed developed exceptional contractile properties that can be compared with those previously observed in vivo in other non-muscle contractile tissues such as the human placenta.


Dengue, a vector-borne infectious disease caused by the dengue virus, has spread through tropical and subtropical regions of the world. All four serotypes of dengue viruses are endemic in the equatorial city state of Singapore, and frequent localised outbreaks occur, sometimes leading to national epidemics. Vector control remains the primary and most effective measure for dengue control and prevention. The objective of this study is to develop a novel framework for producing a spatio-temporal dengue forecast at a neighbourhood level spatial resolution that can be routinely used by Singapore’s government agencies for planning of vector control for best efficiency.The forecasting algorithm uses a mixture of purely spatial, purely temporal and spatio-temporal data to derive dynamic risk maps for dengue transmission. LASSO-based regression was used for the prediction models and separate sub-models were constructed for each forecast window. Data were divided into training and testing sets for out-of-sample validation. Neighbourhoods were categorised as high or low risk based on the forecast number of cases within the cell. The predictive accuracy of the categorisation was measured.Close concordance between the projections and the eventual incidence of dengue were observed. The average Matthew’s correlation coefficient for a classification of the upper risk decile  is similar to the predictive performance at the optimal 30% cut-off. The quality of the spatial predictive algorithm as a classifier shows areas under the curve at all forecast windows being above 0.75 and above 0.80 within the next month.Spatially resolved forecasts of geographically structured diseases like dengue can be obtained at a neighbourhood level in highly urban environments at a precision that is suitable for guiding control efforts. The same method can be adapted to other urban and even rural areas, with appropriate adjustment to the grid size and shape.The online version of this article (10.1186/s12916-018-1108-5) contains supplementary material, which is available to authorized users. Aedes mosquitoes, and in urban areas, primarily by the anthropophilic Aedes aegypti. The total number of dengue infections globally has been estimated to be 390 million per year [Dengue, a vector-borne infectious disease caused by the dengue virus , has spread through tropical and subtropical regions of the world in recent decades [per year , of whicper year . Dengue per year . Howeverper year , making Aedes mosquitoes and transmission of dengue. Since 2013, a dengue incidence of more than 150 per 100,000 population has been reported [In the equatorial city state of Singapore, since the 1990s there has been a dramatic increase in the number of notified dengue cases, and all four serotypes are endemic . Singaporeported . Althougreported , has beereported . The vacreported , but increported , 17.At present, vector control remains the primary and most effective measure for dengue control and prevention . The NatAs well as providing an indication of where dengue transmission is ongoing, incident case data also foreshadow where future outbreaks are most likely, and hence provide a guide to which areas could be prioritised for preventive efforts. To do so requires quantifying the likely number of cases in different areal units, which can be addressed through short-term forecasting.In the literature, various models have been proposed for the prediction of dengue cases. Machine learning methods  have been used at national , sub-natEven within a small city state such as Singapore, spatial variations in risk may be profound, reflecting differences in urban density, the presence of natural areas (such as rainforest and reservoirs) and differential age profiles of different housing estates, and as such, a finer resolution forecast, if one were available, would potentially allow better targeting of the response. The objective of this study is, therefore, to develop a new approach for spatio-temporal dengue forecasting at a finer spatial resolution that can be routinely used by Singapore’s government agencies for planning of vector control for best efficiency, and which may potentially be adapted to other settings.Our objective is to develop a suite of models, each of which will make a forecast for one specified time window, based on the data available at the time the forecast is made. Each model will predict for each neighbourhood the number of cases within a 1-week interval, which will then be used to rank neighbourhoods according to projected risk. This ranking can then be used to identify those areas to be prioritised for interventions, subject to resource availability. Accuracy will be assessed by correlating observed and actual numbers of cases and calculating the receiver operating characteristics when neighbourhoods are classified as high or low risk.The forecasting algorithm uses a mixture of purely spatial, purely temporal and spatio-temporal data to derive dynamic risk maps for dengue transmission.neighbourhoods), from 2010 to 2016, spanning the major residential areas of the country.The Ministry of Health, Singapore, continuously monitors the incidence of dengue through mandatory notification of virologically confirmed or laboratory-confirmed cases. The residential address and date of onset of each case in Singapore are recorded. We aggregated individual-level data into weekly number of cases in 315 spatial units of size 1 km × 1 km  in Singapore. These data were aggregated and used to determine the connectivity between different neighbourhoods, which was subsequently used to derive a variable we called the Aedes mosquito and dengue incidence, this was used as a predictor in the model.Building age was obtained from the Housing Development Board and the Urban Redevelopment Authority and averaged over all buildings within a neighbourhood. Previous studies have shown that the quality of buildings can impact the presence of potential breeding habitats , thus inMeteorological data are incorporated to account for the important role that climate has in the mosquito life cycle. Despite Singapore’s small size, there are some systematic differences in climate across the country , and to Other than weekly incidence in the cells, individual-level dengue incidence data were aggregated into weekly national cases as a proxy for the general epidemic level.The vegetation index refers to the Normalised Difference Vegetation Index (NDVI), which is an index of plant viridescence or photosynthetic activity. NDVI is based on the observation that different surfaces reflect different types of light differently. NDVI data were obtained from the Centre for Remote Imaging, Sensing and Processing in the National University of Singapore from a processed satellite image. Travel history data derived from trips made using EZLink cards (a card to pay for public transport fares in Singapore) were used to measure how connected each neighbourhood is to other parts of the country by public transport. These were processed and aggregated by the provider, prior to analysis, which derived a connectivity ranking based on the number of trips in and out of each cell  squares of size 1 km × 1 km covering residential areas of Singapore were included in each sub-model. For each sub-model, information for all 315 grid neighbourhoods at all time points in the training set were included. Each candidate predictor appeared several times in each sub-model, at different historical lags. To allow for contagion and typical epidemic duration, we used past incidence of up to 8 weeks. To accommodate non-linearities, we also used past incidence squared, cubic, and square root, up to 8 weeks in the past. Polynomials are commonly used to approximate any non-linearity in the relationship between the covariate and outcome, and thus, we allow (but do not force) polynomial terms to account for potential non-linearities between future number of cases and autoregressive terms. In addition, the total number of cases in nearby areas were included at up to 8 weeks lag. Two tiers of nearby areas were used: within 1 km radius and within a ring from 1 km to 2 km from the centroid of the neighbourhood of interest. These are depicted in Additional file k = 1 to 12 weeks), a separate LASSO sub-model was developed, which used data available at the time of the forecast only. Each LASSO sub-model is as follows:yt, i is the number of cases  in neighbourhood i in week t. The terms nt, i, 1 and nt, i, 2 are the total number of cases  from all neighbourhoods whose centroids are within 1 km radius and within a ring from 1 km to 2 km from the centroid of neighbourhood i, in week t, respectively. Wt, i, c represents the climatic variable  at time t in neighbourhood i. Tt, i measures the number of cases moving into neighbourhood i in week t, derived from a one-time telco dataset on the movement of users. At,i measures average building age in neighbourhood i in week t. Nt is the national total number of cases  in week t. Vi and Ui measure the vegetation and connectivity index of neighbourhood i. Detailed information on the type of each set of variables are documented in Additional file z-scored prior to estimation and the coefficients were rescaled afterwards.For each forecast window  for that time point. The choice of dichotomising at 10% was taken considering the operating capacity of the NEA for vector control. Predicted cases during the validation period (2016) constitute a genuine out-of-sample forecast. During the training period (2010–2015), the full time span was used to estimate parameters, but only covariates available at the time of the forecast were used to make the forecast. As such, predictive accuracy may be slightly overstated for the training period.In the model building, predictive accuracy was measured using the root-mean-square error. Subsequently, we assessed the predictive accuracy by evaluating the accuracy of their categorisation of high-risk areas for the validation dataset. For each forecast window, a receiver operating characteristic (ROC) curve —frequently used to evaluate classifiers’ performance—was derived . PredictTo assess the robustness of the findings to the choice of the 10% cut-off we currently adopted for the categorisation, an average Matthew’s correlation coefficient was calculated for each forecast window at 14 different cut-off points . This measures the correlation coefficient between the observed and predicted binary classification, and thus the quality of binary classifications , and takAll statistical analysis were performed using statistical software R .Selected independent variables in the prediction model are presented in Fig. Figure Additional file 13: Video S1. Video of 1-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1177 kb)Additional file 14: Video S2. Video of 2-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1159 kb)Additional file 15: Video S3. Video of 3-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1148 kb)Additional file 16: Video S4. Video of 4-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1125 kb)Additional file 17: Video S5. Video of 5-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1107 kb)Additional file 18: Video S6. Video of 6-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1097 kb)Additional file 19: Video S7. Video of 7-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1071 kb)Additional file 20: Video S8. Video of 8-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1057 kb)Additional file 21: Video S9. Video of 9-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1037 kb)Additional file 22: Video S10. Video of 10-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1019 kb)Additional file 23: Video S11. Video of 11-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 1008 kb)Additional file 24: Video S12. Video of 12-week ahead forecast and actual distribution of dengue incidence in 2016. (MP4 992 kb)The quality of the spatial predictive algorithm as a classifier is measured by ROC curves and the respective AUCs. ROC curves for prediction windows at 1, 2, 4, 8 and 12 weeks are presented in Fig. The effect of risk factors on local dengue risk are shown in Figs. An overall view of the 1-week ahead prediction model is shown in Fig. In Singapore, the average annual economic impact of dengue has been estimated to be around US$100 million, of which 42–59% is attributable to the cost of control . RoutineThus, in this study we developed a novel method to forecast spatial risk within an urban environment at a neighbourhood resolution up to 3 months in advance, using a LASSO-based prediction model. The method gave rather accurate forecasts (AUCs > 0.8 within the next month), with a high correlation with the subsequent incidence data. However, for longer forecast windows, the risk reverted to a baseline risk profile for the neighbourhood. By implementing it as part of our standing vector control programme, the spatio-temporal prediction model can potentially change the current dengue control paradigm into a dengue prevention approach by forecasting dengue risk at a finer resolution in the urbanised environments in which the dengue vectors proliferate. This would allow targeted public health control measures that would use resources most efficiently. The system was robust to changes in the baseline incidence over time  strugglIn conclusion, this report demonstrates that spatially resolved forecasts of geographically structured diseases like dengue can be obtained at a neighbourhood-level in highly urban environments at a precision that is suitable for guiding control efforts.Additional file 1:Supplementary information. (DOCX 19 kb)Additional file 2:Figure S1. Demonstration of the two tiers of neighbouring cells in the study. (PNG 338 kb)Additional file 3:Table S1. Groups of variables and their respective types that are included in the LASSO model. (DOCX 11 kb)Additional file 4:Figure S2.Temporal and spatial average of the weekly number of cases in all grid cells from 2010 to 2016. (PNG 200 kb)Additional file 5:Figure S3. Temporal and spatial average of the sum of the weekly number of cases in all first-tier neighbouring cells (within 1 km) from 2010 to 2016. (PNG 199 kb)Additional file 6:Figure S4. Temporal and spatial average of the sum of the weekly number of cases in all second-tier neighbouring cells (between 1 km and 2 km) from 2010 to 2016. (PNG 198 kb)Additional file 7:Figure S5. Temporal and spatial average of the average temperature in all grid cells from 2010 to 2016. (PNG 202 kb)Additional file 8:Figure S6. Temporal and spatial average of the maximum temperature in all grid cells from 2010 to 2016. (PNG 200 kb)Additional file 9:Figure S7. Temporal and spatial average of the minimum temperature in all grid cells from 2010 to 2016. (PNG 201 kb)Additional file 10:Figure S8. Temporal and spatial average of the average humidity in all grid cells from 2010 to 2016. (PNG 201 kb)Additional file 11:Figure S9. Temporal and spatial average of the movement of incoming incidences in all grid cells from 2010 to 2016. (PNG 198 kb)Additional file 12:Figure S10. Temporal and spatial average of the average building age in all grid cells from 2010 to 2016. (PNG 197 kb)Additional file 25:Table S2. Average Matthew’s correlation coefficient for all 12 forecast windows at 14 different cut-offs. Cut-off are set at different levels so that different percentages of the cells are classified as higher risk areas. (DOCX 12 kb)Additional file 26:Table S3. Summary of parameters included in LASSO forecast model for the 1-week ahead forecast. Table S4. Summary of parameters included in LASSO forecast model for the 2-week ahead forecast. Table S5. Summary of parameters included in LASSO forecast model for the 3-week ahead forecast. Table S6. Summary of parameters included in LASSO forecast model for the 4-week ahead forecast. Table S7. Summary of parameters included in LASSO forecast model for the 5-week ahead forecast. Table S8. Summary of parameters included in LASSO forecast model for the 6-week ahead forecast. Table S9. Summary of parameters included in LASSO forecast model for the 7-week ahead forecast. Table S10. Summary of parameters included in LASSO forecast model for the 8-week ahead forecast. Table S11. Summary of parameters included in LASSO forecast model for the 9-week ahead forecast. Table S12. Summary of parameters included in LASSO forecast model for the 10-week ahead forecast. Table S13. Summary of parameters included in LASSO forecast model for the 11-week ahead forecast. Table S14. Summary of parameters included in LASSO forecast model for the 12-week ahead forecast. (DOCX 37 kb)Additional file 27:Figure S11. Comparisons of forecast and actual scenario for the 2-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 189 kb)Additional file 28:Figure S12. Comparisons of forecast and actual scenario for the 3-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 189 kb)Additional file 29:Figure S13. Comparisons of forecast and actual scenario for the 4-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 189 kb)Additional file 30:Figure S14. Comparisons of forecast and actual scenario for the 5-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 31:Figure S15. Comparisons of forecast and actual scenario for the 6-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 32:Figure S16. Comparisons of forecast and actual scenario for the 7-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 33:Figure S17. Comparisons of forecast and actual scenario for the 8-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 34:Figure S18. Comparisons of forecast and actual scenario for the 9-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 35:Figure S19. Comparisons of forecast and actual scenario for the 10-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 36:Figure S20. Comparisons of forecast and actual scenario for the 11-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)Additional file 37:Figure S21. Comparisons of forecast and actual scenario for the 12-week ahead forecast model. a Actual and predicted yearly total number of cases for all neighbourhoods for both within-sample prediction (blue dots) and out-of-sample prediction (dark red dots). b Average risk over all prediction points (both within-sample and out-of-sample) for the 1-week ahead forecast. (PNG 190 kb)